1. Field of the Invention
This invention relates to a biochemical method for determining the fertilization potential in sperm and, in particular, to a method which utilizes the measurement of particular proteins in spermatozoa which reflect sperm development, maturity, DNA integrity and fertility.
2. Description of Related Art
The testicle serves as site for spermatogenesis, sperm production and sperm maturation. Diagnosis of possible problems in spermatogenesis is normally carried out with the determination of sperm concentrations in the ejaculate. The sperm count fluctuates in all men, but the fluctuations are more detectable in oligospermic (OS) men with sperm counts below the 20xc3x97106 sperm/ml semen of normal or xe2x80x9cnormospermicxe2x80x9d (NS) level. Low sperm concentrations may be a consequence of pituitary problems, deficient hormone levels, testicular disorders and diminished sperm production, retrograde ejaculation, partial obstruction of the ejaculatory tract, age, environmental factors, fever or excess heat, exposure to organic solvents in the work place, etc.
It has been shown that male factor is a contributory cause of infertility in about 40% to 50% of the couples. The overwhelming majority of these infertile and subfertile men are oligospermic and/or asthenospermic (sperm motility is lower than 50%). Management problems exist with respect to these infertile men, as these men may try to father children for years without success. With intrauterine placement of the sperm the pregnancy rates for couples with male factor infertility are only about 15-20% per cycle. This is in spite of the fact that there is no detectable difference in sperm concentrations and motility among the groups of husbands who do or do not cause pregnancy following intrauterine insemination treatment. The pregnancy rates with in vitro fertilization treatment are higher, but adequate sperm concentration or sperm motility does not assure the occurrence of pregnancies. There are also men who suffer from unexplained male infertility, i.e. sperm with diminished fertility in spite of normal sperm concentrations.
Beyond the classical semen analysis parameters, i.e., sperm concentration, motility and velocity, present approaches to evaluate selected sperm functions include the assessment of motile sperm yield following migration or xe2x80x9cswim-upxe2x80x9d; measurements of acrosine activity, i.e., the enzyme which facilitates sperm penetration; the hypoosmotic sperm swelling procedure which probes the integrity of the sperm membrane, the sperm-hemizona assay which tests the ability of sperm to bind to segments of human oocytes, the sperm chromatin structure assay; the human sperm zona-free hamster oocyte penetration test, which is more consistent on the negative side (e.g. penetration rates below 15-20% and diminished success in human in vitro fertilization) than it is a measure of fertility. None of these tests, with the exception of the hemi-zona assay, address the overall physiological soundness of the spermatozoa or showed a high correlation with fertilizing potential or occurrence of pregnancies.
It became increasingly apparent that a new approach was necessary for the assessment of sperm fertility/infertility. This new approach was based on measurement of objective sperm biochemical parameters which were shown to have an essential role in the management of male infertility. Such a method for testing sperm quality and fertilizing potential was developed and disclosed in U.S. Pat. No. 4,945,044, xe2x80x9cObjective Biochemnical Method for Determining Fertilization Potential in Oligospermic Menxe2x80x9d by the inventor of the present application. This method comprised obtaining a sperm sample; detecting the CK enzyme isoforms from the sperm sample, measuring a first CK enzyme concentration for CK-X isoform of the CK enzyme; and determining the sperm quality parameter based upon the first CK enzyme concentration. A second CK enzyme isoform, the CK-B was also measured as a basis for determining the sperm quality parameter. Measurements of the levels of CK-X and CK-B isoforms by electrophoresis and fluorescence visualization technique were used to establish the first and second enzyme concentrations. The sperm quality parameter, as expressed (% CK-X/(CK-X+CK-B)) was proportional to the ratio of the CK-X isoform level to the sum of CK-X and CK-B isoform levels. Sperm fractions which met a predetermined minimum sperm quality level were deemed to be of adequate fertility and selected for use in vivo or in vitro fertilization attempts on oocytes. The validity and predictive value of CK-X/CK-M ratio has been demonstrated in several clinical studies.
Despite the increase in accuracy of the sperm quality parameter described in the ""044 patent, the method of testing for the CK-X isoform of the CK enzyme in sperm requires an electrophoretic analysis which is not readily available outside of laboratories. Cost pressures in health care, including reproductive health care, have encouraged the development of tests which do not require relatively expensive laboratory analysis. Sperm quality tests which could be performed in physicians"" offices, at relatively lower cost and with more readily available equipment, would represent a great advance in fertility treatment.
Bearing in mind the problems and deficiencies of the prior art, it is therefore an object of the present invention to provide an improved method for testing of sperm quality, which also shows high correlation with fertilizing potential and occurrence of pregnancies.
It is another object of the present invention to provide a method and kit for testing sperm quality which may be readily performed outside of a laboratory environment.
A further object of the invention is to provide a sperm quality test which may be performed at lower cost and in physicians"" offices.
Still other objects and advantages of the invention will in part be obvious and will in part be apparent from the specification.
The above and other objects, which will be apparent to those skilled in the art, are achieved in the present invention which provides, in one aspect, a method of testing sperm quality comprising obtaining a sample of sperm to be tested; detecting and measuring amount of testis-specific chaperone protein in the sperm; and determining a sperm quality parameter based upon the amount of chaperone protein, wherein an increased amount of the chaperone protein indicates a higher sperm quality. Preferably, the chaperone protein is detected and measured either by binding one or more antibodies specific to the sperm chaperone protein to the sperm and measuring the antibody content or measuring ATP bound to the sperm chaperone protein. In the case of the latter method, the chaperone protein may be detected and easured by measuring ATP bound to the sperm chaperone protein, and such easuring is by chaperone protein-bound and CK-B generated ATP measurement, or by haperone protein ATP bioluminescence. Preferably, the chaperone protein is detected and measured by a chaperone protein-specific immuno-assay to determine ATP bound to the sperm chaperone protein.
The preferred method further includes detecting and measuring the amount of ATP generated by the CK-B enzyme in the sperm, and comparing the amount of testis-specific chaperone protein detected and measured in the sperm to the amount of ATP bound to CK-B enzyme detected and measured in the sperm. This provides a ratio of sperm containing the testis-specific chaperone protein to sperm CK-B enzyme, which is used to determine the sperm quality parameter. The chaperone protein may be detected and measured by enzymatic determination of the sperm ATP, with and without CK substrate, in order to measure the chaperone protein-bound ATP versus ATP generated by the sperm CK-B, or by chemical measurement of the chaperone protein-bound ATP in sperm.
Any of these detection methods preferably further include measuring the total amount of sperm in the sample and comparing the amount of testis-specific chaperone protein detected and measured in the sperm to the total sperm to provide a concentration of sperm containing the testis-specific chaperone protein which is used to determine the sperm quality parameter. The total amount of sperm in the sample may be measured by measuring sperm concentration, sperm cytoplasmic protein content, total sperm protein content or sperm DNA. In the case of the latter, the total amount of sperm in the sample may be measured by measuring sperm DNA by colorimetry.
Where the amount of chaperone protein is determined by immuno-assay, a minimum sperm quality parameter is preferably determined to be at least about 10 xcexcg per mg of sperm protein, or per 108 sperm. Where the amount of chaperone protein is determined by comparing the amount of ATP bound to the testis-specific chaperone protein in the sperm to the amount of ATP generated by CK-B enzyme in the sperm, a minimum sperm quality parameter is preferably determined to be at least about 10% of ATP bound to the testis-specific chaperone protein as compared to ATP generated by CK-B enzyme. Where the amount of chaperone protein is determined by measuring ATP bound to the sperm chaperone protein, a minimum sperm quality parameter is preferably determined to be at least about 0.2 n M of ATP per mg of sperm protein, or per 108 sperm.
The sperm quality parameter is used to predict one or more of the following: i) sperm fertility independently from sperm concentration in semen; ii) sperm maturity changes in men who are treated with male contraceptive methods, independently from sperm concentration in semen; iii) sperm maturity changes in men who are exposed to reproductive toxicity, independently from sperm concentration in semen; iv) extent of sperm cytoplasmic retention; v) sperm plasma membrane remodeling; vi) ability of the sperm to bind to the zona pellucida of oocytes; vii) sperm morphology; viii) level of sperm lipid peroxidation; ix) sperm DNA integrity or extent of sperm DNA fragmentation; x) frequency of sperm chromosomal aneuploidies; and xi) ability of sperm to maintain viability following cryopreservation and thawing.
In another aspect of the present invention, there is provided a method of screening sperm comprising: obtaining a sample of sperm to be tested; detecting presence of testis-specific chaperone protein in one or more individual sperm in the sample; and segregating the sperm based upon presence of the sperm chaperone protein, wherein presence of the chaperone protein indicates a higher sperm quality. The chaperone protein may be detected by binding one or more antibodies specific to the chaperone protein to the sperm or measuring ATP bound to any sperm chaperone protein.
In one preferred method applicable to humans, the chaperone protein is detected by binding one or more HspA2-specific antibodies to the sperm chaperone protein, labeling the HspA2-specific antibodies to which chromophores are conjugated, and observing the chromophore-immuno-labeled sperm, visually or with automated, computer assisted semen analysis. The chromophore-immuno-labeled sperm may be detected in semen, in sperm fractions, in sperm smears, or in tissues or fluids of the male or female reproductive tract.
Individual ejaculated, epididymal or testicular sperm may be selected based on presence of the sperm chaperone protein, and such sperm containing the chaperone protein are then injected into an egg to fertilize the egg. Alternatively, a sperm fraction may be selected based on presence of the sperm chaperone protein, and such sperm fraction containing the chaperone protein are then used to fertilize an egg.
Yet another aspect of the present invention provides a method of contraception in women comprising: providing sperm; binding one or more antibodies specific to a protein accessible on individual sperm or the sperm tail; and inhibiting movement of sperm to which is bound the one or more antibodies, thereby inhibiting fertilization by the antibody-bound sperm. One or more of the antibodies may be bound to protein accessible on individual sperm tails. Preferably, one or more antibodies are bound to chaperone protein present on plasma membrane of sperm. More preferably, a portion of the sperm contains testis-specific chaperone protein, antibodies specific to the sperm chaperone protein which are bound to individual sperm containing the chaperone protein, leaving individual sperm not containing chaperone protein free of the antibodies, and movement of sperm to which is bound antibodies specific to the sperm chaperone protein is inhibited. Such a method may be conducted by introducing selected epitope peptides of the chaperone protein into the female reproductive tract for the systemic generation of chaperone protein antibodies to arrest sperm motility, by introducing into the reproductive tract anti-chaperone protein antibodies in order to diminish sperm motility and fertilization potential, or by introducing into the female reproductive tract sperm barriers containing an active component of anti-chaperone protein antibodies or other antibodies directed to sperm plasma membrane proteins which are able to capture and arrest sperm and diminish fertilization potential. The sperm barriers may be intrauterine or intracervical devices.
In all of the above methods, where the sperm is human sperm, the chaperone protein is preferably HspA2. Where the sperm is non-human sperm, the chaperone protein is a variant testis-specific chaperone protein.
A further aspect of the present invention provides a multi-part system for determining the quality or fertilization potential of a sperm sample by detecting and measuring amount of testis-specific chaperone protein in the sperm, wherein an increased amount of the chaperone protein indicates a higher chance of fertilization by the sperm. In one embodiment the system comprises a sperm washing solution; a sperm homogenization solution; a composition containing an antibody specific to the sperm chaperone protein; and a composition capable of visually distinguishing any of the antibody which is bound to the sperm chaperone protein. In another embodiment, the system comprises a sperm washing solution; a sperm homogenization solution; a composition adapted to detect ATP bound to the sperm chaperone protein; and a composition capable of visually distinguishing any of the ATP which is bound to the sperm chaperone protein.
The Testis-Specific Chaperone Protein
The present invention provides an improvement to the method disclosed in the aforementioned U.S. Pat. No. 4,945,044, the disclosure of which is hereby incorporated by reference. An objective biochemical method is provided based on the measurement of the testis-specific chaperone protein (xe2x80x9cCPxe2x80x9d). In humans, the testis-specific chaperone protein is believed to be HspA2 testis-specific chaperone protein. In rodents, the homologue testis-specific chaperone or heat-shock protein is believed to be HSP70-2 chaperone protein. The chaperone protein is also present in the sperm of other species, such as in horse sperm, and its particular identity may be determined without undue experimentation. The terms xe2x80x9cchaperone proteinxe2x80x9d or xe2x80x9cCPxe2x80x9d as used herein also includes variants of the protein.
As a result of testing of 1- and 2-dimensional SDS-polyacrylamide gel electrophoresis, amino acid sequencing, immunocytochemistry of the testicular tissue and of mature sperm, it has unexpectedly been determined that the CK-X isoform comprises the aforementioned chaperone protein, e.g., HspA2 in men, as a developmentally regulated marker of sperm maturity and function. The present invention is based on the discovery that the chaperone protein biochemical marker, HspA2 in men, reflects the cellular maturity of spermatozoa and provide a method of monitoring sperm maturation. In rodents the importance of HSP70-2 has been demonstrated by the selective destruction of the HSP70-2 gene. The HSP70-2 knock-out mice showed diminished sperm concentrations and increased sperm DNA degradation.
It has also been found that the electrophoretic properties of HspA2 are similar to those of the muscle CK-M on the native Agarose gels of the type used for CK isoform analysis. Moreover, the assay system for CK activity also detects adenosine triphosphate (ATP) synthesis, so that the ATP which binds the HspA2 chaperone may be perceived as new ATP produced. The fact that the chaperone protein has bound ATP is important from the point of view of sperm chaperone protein ratio measurements because this ATP can be measured and distinguished from the ATP which is generated by the sperm CK-B in the presence of the creatine phosphate and ADP substrates.
Detecting and Measuring Amount of Testis-Specific Chaperone Protein
In addition to determining that testis-specific chaperone protein is important in determining sperm quality, the present invention provides a method for testing sperm maturity by detecting the levels of chaperone protein, and determining the chaperone protein ratio, in sperm samples.
There are several approaches for the measurement of chaperone protein ratio, which is a measure of the amount of sperm chaperone protein versus sperm mass of the sample. Assessment of sperm mass may be based on sperm concentrations, sperm CK-B or other sperm cytoplasmic protein, total sperm protein, sperm DNA content or other factors. Three approaches have been preferably identified for chaperone protein assessment: i) immuno assay with chaperone protein antibodies; ii) enzymatic determination of the sperm ATP, with and without CK substrate, in order to measure the chaperone protein-bound ATP versus ATP generated by the sperm CK-B, and iii) chemical measurements of the chaperone protein-bound ATP in sperm. The first approach determines the amount of chaperone protein by binding one or more antibodies specific to the sperm chaperone protein to the sperm and measuring the antibody content. The second and third approaches measure ATP bound to the sperm chaperone protein.
The steps of the chaperone protein ratio assessment preferably include: 1) measurement of sperm chaperone protein concentrations; 2) determination of sperm concentrations or sperm mass using CK-B, another cytoplasmic protein, total sperm protein, sperm DNA or other factors that reflect sperm numbers in the test aliquot; and, finally, 3) using two sets of data as a basis for determining the chaperone protein ratio sperm maturity parameter. Measurements of the levels of chaperone protein and sperm mass may be carried out by computer assisted semen analysis, by immuno assay and fluorescence techniques, by the ATP associated with chaperone protein and ATP generated by the CK-B, respectively, or by colorimetry, immunocytochemistry or other immuno-techniques, enzymatic measurements, fluorescent labeling and cell sorting.
The method for the detection of sperm chaperone protein concentrations and chaperone protein ratios or the ratios of CP/sperm concentrations, CP/sperm cytoplasmic proteins or CP/sperm DNA in semen is composed of the phases of the chaperone protein determination and of the denominator factors of sperm concentrations, concentrations of CK-B or other cytoplasmic proteins, total sperm protein, or sperm DNA content. The detection of the chaperone protein may be carried out by immunoassay, by an enzymatic approach and by a chemical method of ATP detection using the luciferase bioluminescence assay. These varieties of methods are all aimed at the determination of the expression level CP/(CK-B+CP) which is the measure of sperm maturity and fertilizing potential as described above.
Proper specimen collections and washing of the sperm for removal of seminal fluid is important to obtain an accurate interpretation of chaperone protein and sperm mass analysis. If the determination is not carried out immediately after collection, the washed sperm pellet covered with a buffer containing an xe2x80x94SH group protectant should be refrigerated and it may be stored at 4xc2x0 C. Alternatively, for long-term storage, the sperm pellets may be kept at xe2x88x9270xc2x0 C. Repeated freezing and thawing destroys protein structure and should be avoided.
The determination of chaperone protein ratio may also be accomplished by the chemical measurements, for instance with the luciferase which may measure chaperone protein-bound ATP, after a metabolic inhibitor is added to the sperm and to the sperm extract in order to inhibit the de novo ATP production. Alternatively, the chemiluminescent detection may be performed following electrophoretic separation of the chaperone protein-ATP complex and of the CK-B. The detection and scanning, as well as the integration of the signals, are now automated and readily available as workstations.
For the detection of chaperone protein and CK-B, which represents the sperm mass, one can also use an enzymatic method. This is based on the fact that the chaperone protein-bound ATP may be directly detected by the NAD/NADH system, whereas ATP generation by the CK-B will occur only in the presence of the CK-B substrates, ADP and creatine phosphate. Thus, the difference between ATP measured in the sperm extracts in the absence and presence of the substrates will provide the concentrations of the two factors necessary for the chaperone protein ratio measurement, chaperone protein and CK-B. The detection may occur in microplates, slides or on Agarose gels which are compatible with plate or slide readers. The sperm proteins may be spotted in two wells, or they can be separated by electrophoresis in an agarose plate or other medium.
For the detection of the total sperm ATP (comprised of the chaperone protein-bound and CK-B generated ATP), the sperm extract is overlaid with the ATP detection substrate. CK catalyzes the transfer of phosphate from creatine phosphate to an adenosine diphosphate (ADP) forming creatine and adenosine triphosphate (ATP). The ATP formed in the CK reaction is used to produce glucose-6-phosphate. This reaction is catalyzed by the enzyme hexokinase. The glucose-6-phosphate is then oxidized by the enzyme, glucose-6-phosphate dehydrogenase with a simultaneous reduction of nicotinamide adenine dinucleotide (NAD) to the fluorescent derivative, NADH. The NADH fluorescence, which is proportional to the presence of chaperone protein-bound plus CK-B generated ATP, is quantified on agarose plates by a scanning fluorometer. In measurement of the ATP bound to chaperone protein alone, the ATP is directly available for glucose-6-phosphate synthesis, leading to production of the respective NADH fluorescence signal which represents the chaperone protein concentration alone, and may be deducted from the total ATP signal to provide the ATP generated by the CK-B. The amounts of chaperone protein-bound and CK-B generated ATP are proportional to the content of these proteins in the sperm extract and provide the chaperone protein ratio expressed as %CP/(CP+CK-B).
In addition to the chaperone protein measurement in sperm extracts, due to the development of chaperone protein specific antibodies, one can detect chaperone protein expression in individual mature sperm by chaperone protein-specific antibodies, which allows the assessment of the proportion of mature sperm in semen samples of sperm fractions tested, or in tissue or body fluid extracts. This method can be also adapted as a feature for a computer assisted semen analyzer which then in addition to sperm concentration, motility, velocity and other parameters will provide the percent of mature sperm in a specimen.